Flow cytometric analysis of aortic immune cell composition

For flow cytometric analysis of aortic immune cells, the mice were euthanised after 25 weeks of treatment. Blood was collected from the heart and anticoagulated with 1 mmol/l EDTA. The analysis of the aorta was performed according to the protocol detailed by Butcher et al with slight modifications [19, 24]. Single cells were stained with the LIVE/DEAD Fixable Aqua Dead cell stain kit (Invitrogen Life Technologies, CA, USA) to exclude dead cells. To prevent unspecific binding, the cells were incubated with anti-CD16/32-antibody (clone93) before antibody staining with the following antibodies for detecting macrophages: F4/80-AF488 (cloneBM8), CD45-PE (clone30-F11), CD11b-PacBl (cloneM1/70). Unless otherwise indicated, antibodies were purchased from Biolegend. Absolute cell concentrations were determined using Flow-Count Fluorospheres (Beckman Coulter) according to the manufacturer’s instructions. In the case of lymphocyte analysis, erythrocytes were lysed with hypotonic ammonium chloride solution followed by centrifugation at 300 g for 10 min and 4°C. The cells were then resuspended in PBS containing 2 mmol/l EDTA and 0.5% BSA protein extraction buffer for staining. For the detection of neutrophils and monocytes including subsets, the blood samples were stained before lysis of erythrocytes using antibodies against the following surface molecules: Ly6C-AF488 (cloneHK1.4; Biolegend), CD11b-PE (cloneM1/70; BD Biosciences), CD115-APC (cloneAFS98; eBioscience, San Diego, CA, USA) and Ly6G-PacBl (clone1A8; Biolegend). Lymphocytes were separated with antibodies against the following surface molecules: CD4-FITC (cloneRM4-5, Invitrogen), CD45-PE (clone30-F11, Biolegend), CD8a-AF647 (clone53-6.7, Biolegend), CD3-APC/Cy7 (clone17A2, Biolegend), CD19-PacBl (clone6D5, Biolegend). To prevent unspecific binding, the cells were incubated with anti-CD16/32-antibody (clone93; Biolegend) before antibody staining. The samples were measured on Gallios flow cytometer (Beckman Coulter). The data were analysed using Kaluza flow analysis software (Beckman Coulter).

Murine blood cells counts after 8 and 25 weeks of dapagliflozin are given in Electronic supplementary material (ESM) Tables 5 and 6.